Dissertations / Theses on the topic 'Human cell culture'

In human body, the most common structures formed by epithelial cells are hollow cysts or tubules. The key feature of the cysts and tubules is the central lumen, which is lined by epithelial cell sheets. The central lumen allows material exchange, thus it is indispensable for the proper function of the epithelial tissue. In order to understand the way that the epithelial cells form highly specialized structure, an in vitro three-dimensional (3D) culture system was established. The Caco-2 cells were embedded in reconstituted basement membrane termed matrigel, whose biochemical constitution and physical properties were similar with the in vivo environment. The Caco-2 cells in matrigel spontaneously formed spherical multi-cell cysts, which could continuously expand. The confocal imaging and reconstruction technique helped understand the cyst structure and its formation process. The cysts developed central lumen surrounded by a layer of polarized cells. The apical domain of the cells faced the lumen, while the basal domain attached to the extracellular matrix. In the mature cysts, fluid was secreted by the cells around the lumen at the apical domain, and accumulated in the central lumen. The laser burning experiment showed that the intraluminal pressure was higher than the outer environment. The intact cell sheet was required to keep the engorged morphology of the cysts. The tension of the cell layer balanced with the intraluminal pressure. To investigate the effect of pressure on cyst development, the cysts were treated with cholera toxin, which could increase intraluminal pressure through promoting apical secretion. The time-lapse images showed that under cholera toxin treatment, the expansion of the cysts was accelerated. The high intraluminal pressure led to shape change of thecells, followed by increase in cell proliferation rate. Cholera toxin itself could not promote cell growth. In the3D cultured cysts, it was the increased intraluminal pressure that directly induced the acceleration of cell proliferation. It indicated that not only biochemical signals, but also mechanical force, contributed to epithelial morphogenesis. The mechanical stimulation could be converted into biochemical signals, further affect cell behavior. In response to mechanical stimulation, the focal adhesion kinase was activated in the cells around the cyst lumen. Furthermore, the microarray analysis suggested that multiple signaling pathways were altered under intraluminal pressure stimulation, including the pathways related to cytoskeleton organization, cell cycle and cell adhesion. Taken together, comparing with the conventional two-dimensional cell culture on rigid surface, the three-dimensional culture system provided the cells a more physiological environment. The 3D culture system allows the epithelial cells to form well-organized hollow structure. It is a convenient model for investigating the process and mechanism of epithelial morphogenesis.
published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy

Cornea is the front transparent window of the eye which is responsible for optimal and clear vision. Transparency of this tissue is highly inevitable and cannot be compromised. Human cornea is made up of multiple layers out of which the posterior layer ‘endothelium’ is responsible for the transparency of the cornea. Endothelium is a monolayer of cells that allow the ions and solutes to transport from aqueous humour to the cornea and back which in turn maintains the transparency of the cornea by preserving the homeostasis between the anterior and posterior cornea. Earlier, it was observed that the endothelium had non regenerating capability however; recent studies have shown that these cells could be proliferated in vitro. Currently, the only method of treatment is the replacement of the diseased endothelium with the healthy donor endothelium. Penetrating keratoplasty which transplants a full thickness cornea was the only solution a decade ago. However, with the new advancements in the field of corneal transplants, specific surgical techniques like DMEK and DSAEK which replace only a part of the cornea have been identified. DSAEK replaces a part of the stroma along with the Descemet’s membrane and endothelium whereas DMEK only replaces the Descemet’s membrane and the endothelium and does not involve stroma. The results in terms of visual rehabilitation and outcomes have been found to be advantageous in these specific surgical procedures. However, DMEK is more challenging then DSAEK as DMEK is not yet a widespread technique, associated with steep learning curves and difficult donor tissue preparation. Despite DMEK is a challenging procedure it is becoming more popular because of the significant advantages in term of faster visual recovery, less postoperative astigmatism and reduced risk of transplant rejection, as compared to the other EK procedures. DMEK has several advantages in terms of rehabilitation rate and post-operative visual outcomes and therefore it is necessary to further refine this technique for a higher uptake of such surgeries and also considering that this is the only possible treatment for treating the patients suffering from endothelial dysfunctions. Although the corneal transplantation is well advanced, due to a limited supply of donor corneas for the transplantation purposes, alternative approaches like culturing corneal endothelium in vitro play an important role. Culturing the endothelium is not the only problem in EK but transplanting a 20 micron thick graft inside the recipient eye is another challenge. Moreover, the donor availability for culturing the corneal endothelium is less, making this strategy further more complicated. The thesis is therefore structured to highlight two significantly important issues in current scenario of endothelial keratoplasty, 1) posterior corneal transplantation or EK which is the on-going method of treatment for EK and 2) Human corneal endothelial cell culture which is the future of EK. Chapter 1 is an introduction to the world of eye banking, its current nature and development in the modern world and as a support to the surgeons not only in terms of new techniques but also devices for selective surgeries. It also highlights the preservation of the corneal tissues which is an important element in the field of eye banking. Eye banks play a significant role in the field of corneal transplants as they collect the human corneas and process them for transplantation. The corneas that are rejected for transplantation can be used for research and therefore development of eye banking and its research can change the field of corneal transplantation. Chapter 2 introduces the field of corneal cell culture and current techniques that are followed for culturing and possible transplantation of the cultured cells. To understand the reason and requirement of tissue engineering, it is important to study the human cornea, its extracellular matrix and its behaviour in different media. The biomechanical behaviour of the thin tissue i.e. the DM in different conditions becomes a relevant part of this study for future engineering which is studied in chapter 3. It is also important to standardize the currently available treatment options to reduce the burden of endothelial compromised patients in the future and avoid damages or tissue wastage that is currently occurring in the surgical theatres by providing standardized tissues in validated preservation medium which is studied in chapter 4. DMEK promises to become a more popular technique for the replacement of unhealthy corneal endothelium as it shows advantages like early rehabilitation rate and visual outcomes. Chapter 5 highlights the importance of new technique in rolling the DMEK tissue for easy insertion and unfolding in the recipient eye compared to the currently used technique with endothelium rolled in opposite direction. Presently, the DMEK tissues are either prepared in the surgical theatre or are stripped in the eye bank and shipped to the surgeons. However, there is no standardized procedure that could help validate a graft before surgery and provide a ready-to-use graft to the surgeons. Chapter 6 describes about a new technique of pre-loading a graft in a commercially available IOL cartridge which can be used as a preservation, transportation and transplantation device. This technique will further reduce graft wastage and will provide the surgeons a pre-validated graft further reducing the overall time in the surgical theatre and related costs. Thus different approaches for standardizing the DMEK technique were studied in the first phase of the thesis. HCECs are currently being cultured using young donor corneas. There are two major issues, firstly, the availability of the young donor corneas is less compared to the old donor corneas and secondly, there is no standard method of culturing the HCECs obtained so far. Therefore, to reduce the global tissue demand, there is a strong need to culture the HCECs from the old donor corneas which are less proliferative and less robust in nature but with high availability of the donor source. Chapter 7 is a study on isolation of HCECs and further culture of these cells from old donor corneas. Once the protocol was obtained, a full length study was performed with high sample size to prove the consistency of this technique which is highlighted in chapter 8. Meanwhile it was also noted that cells from old donors can be cultured using ROCK inhibitor in combination with Hyaluronic Acid (HA). HA induces mechanical force to the cells attaching them forcefully on the base and allows a higher proliferation of old donor cells which was studied in chapter 9. The second part of the thesis therefore investigates the culturing technique of HCECs from old donor corneas. However, once the cells are cultured, another challenge is to transplant them in the anterior chamber of the eye. This can be performed using two strategies, first, to implant the cells as suspension in the anterior chamber which is already been proposed, but the clinical evidence is still not confirmed yet, and second, to develop a carrier to transport the cultured cells. In chapter 10, we identified fish scales as a great source of collagen and therefore have investigated it as a potential scaffold to be used for HCECs culture and transplant in the future. It is also important to understand the regulations that govern the scientific studies and its use for clinical applications. Therefore, we also identified rHSA as a source to replace FCS for preserving human corneas in chapter 11. This will also help to create a synthetic media that could be used for GMP purposes for HCECs culture in the future. In conclusion, it was observed that pre-loading the tissues with endothelium-flapped inwards and preserved in dextran based medium could be a potential solution for providing a validated and standardized DMEK graft for the treatment of current endothelial dysfunction. Eye banks play a major role in the development of these surgical techniques and related devices which will change the face of corneal transplantation in the future. Alternatives like HCECs culture has a potential for the treatment of endothelial disorders and carriers like FSS could be used for culturing and transplanting these cells. However, the efficacy of these cells will only be validated after the clinical study. Considering the regulatory issues, synthetic medium would help both, the eye banks for preserving the corneas and its new products like pre-loaded DMEK and for cell culture in the future.
La cornea è quel tessuto trasparente che riveste la superficie anteriore dell'occhio, e che consente di avere una visione ottimale e chiara. La trasparenza di questo tessuto è fondamentale e non può essere compromessa. La cornea umana è costituita da più strati,tra cui lo strato posteriore o “endotelio” è responsabile della trasparenza della cornea. L’ endotelio è un monostrato di cellule che permettono agli ioni ed ai soluti di essere trasportati dall’ umor acqueo alla cornea e viceversa, e che a sua volta mantiene la trasparenza della cornea conservando l'omeostasi tra la cornea anteriore e posteriore. L’endotelio non possiede capacità rigenerative. Attualmente, l'unico metodo di trattamento è la sostituzione dell'endotelio danneggiato con l'endotelio di un donatore sano. La cheratoplastica perforante, che prevede trapianti di cornea a tutto spessore,rappresentava l'unica soluzione terapeuticafino ad un decennio fa. Tuttavia, con i nuovi progressi nel campo dei trapianti di cornea, sono state identificate specifiche tecniche chirurgiche, come DMEK e DSAEK, che sostituiscono solo una parte (o uno strato) della cornea. Sono I risultati ottenuti, in termini di riabilitazione visiva, si sono rivelati vantaggiosi grazie all’utilizzo di queste procedure chirurgiche specifiche. Tuttavia, la DMEK è più impegnativarispetto alla DSAEK in quanto non è ancora completamente standardizzata. La DMEK ha diversi vantaggi in termini di tasso di riabilitazione e risultati visivi post-operatori e quindi è necessario standardizzare questa tecnica per una maggiore diffusione di tali interventi e anche considerando che questo è l'unico trattamento possibile per la cura di pazienti affetti da disfunzioni endoteliali. Sebbene il trapianto di cornea sia in fase avanzata, a causa di una quantità limitata di cornee da donatori ai fini di trapianto, approcci alternativi come la coltura di endotelio corneale in vitro svolgono un ruolo importante. La coltura di endotelio non è l'unico problema nel trapianto di endotelio (EK)dal momento che trapiantare un innesto di 20 micron di spessore all'interno dell'occhio destinatario rappresenta una sfida ulteriore. Inoltre, la disponibilità dei donatori per la coltura di endotelio corneale è inferiore, rendendo questa strategia ulteriormente più complicata. La tesi è quindi strutturata in modo da mettere in evidenza due questioni molto importanti nell’ attuale scenario della cheratoplastica endoteliale, 1) trapianto di cornea posteriore o EK, che è l'attuale metodo di trattamento per la cheratoplastica endoteliale e 2) coltura delle cellule endoteliali della cornea umana, che rappresenta il futuro della cheratoplastica endoteliale. Il Capitolo 1 è un'introduzione sul mondo dell’ Eye Banking, sulle sue caratteristiche attuali, sullo sviluppo nel mondo moderno e sul supporto per i chirurghi, non solo in termini di nuove tecniche, ma anche di dispositivi per interventi selettivi. Si evidenzia anche la conservazione dei tessuti corneali, che è un elemento importante nel campo dell’Eye Banking. Le banche degli occhi svolgono un ruolo significativo nel settore dei trapianti di cornea, dal momento cheraccolgono le cornee umane e le analizzano per ilsuccessivo trapianto. Le cornee non idonee per il trapianto possono essere utilizzate per la ricerca e quindi lo sviluppo dell’Eye Bankinge la ricerca possono influenzare il campo del trapianto di cornea. Il Capitolo 2 introduce l’argomento delle colture cellulari corneali e le tecniche attuali che sono utilizzate per la coltura ed il trapianto di cellule coltivate. Per capire il motivo e l'esigenza dell’ingegnerizzazione dei tessuti, è importante studiare la cornea umana, la sua matrice extracellulare ed il suo comportamento in diversi mezzi di coltura. Il comportamento biomeccanico di un tessuto sottile (DM) in condizioni diverse rappresenta una parte rilevante di questo studio per la futura ingegnerizzazione,che viene descritta nel Capitolo 3. E’ inoltre importante standardizzare il trattamento attualmente disponibile allo scopo di ridurre in futuro l’onere di pazienti con endotelio compromesso ed evitare danni o sprechi di tessuto, che attualmente avvengono nelle sale chirurgiche, fornendo tessuti standardizzati in terreni di conservazione validati, come descritto nel Capitolo 4. La DMEK è considerata il futuro della cheratoplastica endoteliale, dal momento che presenta vantaggi quali la velocità dei tempi di riabilitazione ed i risultati visivi. Il Capitolo 5 mette in evidenza l'importanza della nuova tecnica che consiste nell’arrotolare il tessuto DMEK per consentire un facile inserimento per poi dispiegarlo nell'occhio ricevente, rispetto alla tecnica attualmente utilizzata con endotelio arrotolato in senso opposto. Attualmente, i tessuti DMEK sono o preparati in sala operatoria o allestiti in Banca degli Occhi e spediti ai chirurghi. Tuttavia, non vi è alcuna procedura standardizzata che possa contribuire ad ottenereun lembo endoteliale validato prima dell'intervento e fornire un innesto ready-to-use ai chirurghi. Il Capitolo 6 descrive una nuova tecnica di pre-caricamento di un lembo endoteliale in una cartuccia IOL disponibile in commercio che può essere utilizzato come dispositivo di conservazione, trasporto e trapianto. Questa tecnica consentirà di ridurre ulteriormente gli sprechi nei trapianti e fornirà ai chirurghi un innesto pre-convalidato,riducendo ulteriormente il tempo complessivo in sala operatoria edi relativi costi. Quindi nella prima fase della tesi, sono stati analizzati i diversi approcci per standardizzare la tecnica DMEK. Le HCECs sono attualmente coltivate usando cornee di donatori giovani. Ci sono due aspetti importanti, in primo luogo la disponibilità di tessuti di donatori giovani è minore rispetto a quella di donatori anziani, ed in secondo luogo non vi è, ad oggi, alcun metodo standardizzato di coltura delle HCECs. Pertanto, per ridurre la domanda di tessuti a livello mondiale, vi è una forte necessità di coltivare leHCECsderivanti da cornee di donatori anziani, che sono meno proliferative e meno resistenti in natura, ma per le quali vi è una elevata disponibilità della fonte donatrice. Il Capitolo 7 descrivelo studio sull'isolamento delle HCECs e la successiva coltura di tali cellule ottenute da cornee di donatori anziani. Una volta stabilito il protocollo, è stato eseguito uno studio completocon un alto campionamento, per dimostrare la coerenza di questa tecnica,come evidenziato nel Capitolo 8. Nel frattempo si è anche osservato che le cellule da donatori anziani possono essere coltivate utilizzando l’inibitore ROCK in combinazione con acido ialuronico (HA). HA induce una forza meccanica alle cellule per far sì che siano saldamente attaccate alla base e consentire così una maggiore proliferazione,come descritto nel Capitolo 9. La seconda parte della tesi indaga quindi la tecnica di coltura delle HCECs da cornee di donatori anziani. Tuttavia, una volta che le cellule sono coltivate, un'altra sfida è trapiantarle nella camera anteriore dell'occhio. Ciò può essere eseguito utilizzando due strategie: la prima è quella di ad impiantare le cellule in forma di sospensione nella camera anteriore, tecnica che è già stata proposta, ma che non ha ancora fornito un’evidenza clinica; la secondaè quella di sviluppare un substrato per il trasporto delle cellule coltivate. Nel Capitolo 10, si identifica la colla di pesce (FSS)come una grande fonte di collagene e quindi come un potenziale scaffold da utilizzare per la cultura HCECs e successivo trapianto. E’ inoltre importante capire le norme che regolano gli studi scientifici ed il loro uso nelle applicazioni cliniche. Pertanto, nel Capitolo 11, viene descritta l’identificazione dell’ rHSA come sostitutodell’ FCS per la conservazione di cornee umane. Questo contribuirà anche a creare un terreno di coltura sintetico che potrebbe essere utilizzato per la cultura HCECs in condizioni GMP in futuro. In conclusione, si è osservato che il pre-caricamento di tessuti con endotelio rivolto verso l'interno e conservati in un terreno con destrano, potrebbe rappresentare una possibile soluzione per fornire un lembo per DMEK validato e standardizzato per il trattamento delle disfunzioni endoteliali. Le banche degli occhi svolgono un ruolo importante nello sviluppo di queste tecniche chirurgiche e relativi dispositivi, che potranno cambiarele modalità del trapianto di cornea in futuro. Una tecnica alternativa come la coltura di HCECs ha in sèil potenziale per il trattamento di disturbi endoteliali e substrati come FSS potrebbero essere utilizzati per la coltura edil trapianto di queste cellule. Tuttavia, l'efficacia di queste cellule potrà essere validata solo dopo uno studio clinico. Considerando le questioni regolatorie, il terreno sintetico potrebbe aiutare le banche degli occhi sia per la conservazione delle cornee e dei i nuovi prodotti come DMEK pre-caricati sia, in futuro, per le colture.

The eukaryotic cell cycle orchestrates the precise duplication and distribution of the genetic material, cytoplasm and membranes to daughter cells. In multicellular eukaryotes, cell cycle regulation also governs various organisatorial processes ranging from gametogenesis over multicellular development to tissue formation and repair. Consequently, defects in cell cycle regulation provoke a variety of human cancers. A global view of genes and pathways governing the human cell cycle would advance many research areas and may also deliver novel cancer targets. Therefore this work aimed on the genome-wide identification and systematic characterisation of genes required for cell cycle progression in human cells. I developed a highly specific and efficient RNA interference (RNAi) technology to realize the potential of RNAi for genome-wide screening of the genes essential for cell cycle progression in human tissue culture cells. This approach is based on the large-scale enzymatic digestion of long dsRNAs for the rapid and cost-efficient generation of libraries of highly complex pools of endoribonuclease-prepared siRNAs (esiRNAs). The analysis of the silencing efficiency and specificity of esiRNAs and siRNAs revealed that esiRNAs are as efficient for mRNA degradation as chemically synthesized siRNA designed with state-of-the-art design algorithms, while exhibiting a markedly reduced number of off-target effects. After demonstrating the effectiveness of this approach in a proof-of-concept study, I screened a genome-wide esiRNA library and used three assays to generate a quantitative and reproducible multi-parameter profile for the 1389 identified genes. The resulting phenotypic signatures were used to assign novel cell cycle functions to genes by combining hierarchical clustering, bioinformatics and proteomic data mining. This global perspective on gene functions in the human cell cycle presents a framework for the systematic documentation necessary for the understanding of cell cycle progression and its misregulation in diseases. The identification of novel genes with a role in human cell cycle progression is a starting point for an in-depth analysis of their specific functions, which requires the validation of the observed RNAi phenotype by genetic rescue, the study of the subcellular localisation and the identification of interaction partners of the expressed protein. One strategy to achieve these experimental goals is the expression of RNAi resistant and/or tagged transgenes. A major obstacle for transgenesis in mammalian tissue culture cells is the lack of efficient homologous recombination limiting the use of cultured mammalian cells as a real genetic system like yeast. I developed a technology circumventing this problem by expressing an orthologous gene from a closely related species including its regulatory sequences carried on a bacterial artificial chromosome (BAC). This technology allows physiological expression of the transgene, which cannot be achieved with conventional cDNA expression constructs. The use of the orthologous gene from a closely related species confers RNAi resistance to the transgene allowing the depletion of the endogenous gene by RNAi. Thus, this technology mimics homologous recombination by replacing an endogenous gene with a transgene while maintaining normal gene expression. In combination with recombineering strategies this technology is useful for RNAi rescue experiments, protein localisation and the identification of protein interaction partners in mammalian tissue culture cells. In summary, this thesis presents a major technical advance for large-scale functional genomic studies in mammalian tissue culture cells and provides novel insights into various aspects of cell cycle progression. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 217 MB: Movies, Rohdaten - Nutzung: Referat Informationsvermittlung der SLUB)

Abstract Resistin was discovered in the year 2000. In the mouse, it was reported to be produced by adipocytes and to be linked with insulin resistance and obesity. Human resistin has been shown to be produced by leucocytes but the literature contains contradictory results on its association with obesity and type 2 diabetes. Aim of this study was to clarify the role of resistin in the human context especially in type 2 diabetes and atherosclerosis. The first study examined the possible association of human resistin gene variants with type 2 diabetes. The studied three variants were not associated with type 2 diabetes though they were demonstrated in the second study to be associated with the plasma resistin concentration. However, two gene variants were associated with the prevalence of cerebrovascular disease in subjects with type 2 diabetes. In the third work, the association of the plasma resistin level with the risk factors of atherosclerosis and early atherosclerosis measured with carotid artery intima-media thickness (IMT) were studied. Plasma resistin level was not associated with IMT independently from the known risk factors of atherosclerosis. However, resistin was associated with inflammatory markers highly sensitive CRP and the number of leucocytes whereas insulin resistance did not associate with resistin. In the fourth study, resistin expression in different leucocytes and its modulation, as well as the effect of resistin on monocyte adhesion to endothelium were evaluated. The novel discovery was that resistin is expressed in all the main leucocyte lineages, with monocytes and neutrophils exhibiting the highest relative mRNA and protein levels of resistin. Resistin expression was up-regulated by pro-inflammatory factors in the cells of both innate and adaptive immunity. The present results demonstrating that resistin increases adhesion and expression of some adhesion molecules support the hypothesis that resistin may be involved in the development of atherosclerosis. The above results indicate that resistin is widely produced by leucocytes and therefore may participate in inflammatory processes. Since it may be considered as an inflammatory cytokine, resistin may also influence the development of atherosclerosis. In the future, resistin could possibly be used as a marker of inflammation
Tiivistelmä Vereen erittyvä uusi hormoni, resistiini, löydettiin vuonna 2000. Hiirellä resistiinin on havaittu erittyvän rasvasoluista ja sen on arveltu toimivan linkkinä lihavuuden ja insuliiniresistenssin välillä. Ihmisellä resistiinin tehtävä on toistaiseksi huomattavasti epäselvempi ja toisin kuin hiirellä ihmisen resistiinin korkein ilmentymistaso on valkosoluissa. Tämän väitöstutkimuksen tarkoituksena oli selvittää ihmisen resistiinin toimintaa ja erityisesti sen liittymäkohtia tyypin 2 diabetekseen ja ateroskleroosiin. Ensimmäisessä osatyössä on selvitetty resistiini-geenin nukleotidimuuntelun yhteyttä tyypin 2 diabetekseen ja siihen liittyviin tekijöihin diabetespotilasaineistossa. Resistiinin geenimuuntelu ei tulosten perusteella ole yhteydessä tyypin 2 diabetekseen, vaikka sillä näyttääkin olevan vaikutusta plasman resistiini-pitoisuuteen, mikä havaittiin toisessa osatyössä. Geenimuuntelulla oli havaittavissa yhteyttä aivovaltimosairauteen. Kolmannessa osatyössä plasman resistiinipitoisuuden yhteyttä valtimonkovettumatautiin ja sen riskitekijöihin tutkittiin Pohjois-Pohjanmaalta kerätyssä aineistossa (n = 525). Plasman resistiinipitoisuudella ei havaittu itsenäistä yhteyttä kaulavaltimonseinämänpaksuuteen, joka kuvastaa alkuvaiheen valtimokovettumataudin tasoa. Tulehdukselliset merkit kuten veren valkosolujen määrä ja plasman CRP-pitoisuus liittyivät suurentuneeseen plasman resistiinipitoisuuteen mutta insuliiniresistenssimuuttujat eivät. Neljännessä osatyössä tutkittiin resistiinin ilmentymistä. Resistiinin havaittiin ilmentyvän kaikissa keskeisissä valkosolutyypeissä ja lisäksi tulehdustekijät lisäsivät sen tuottoa. Erityisesti neutrofiileissä ja monosyyteissä resistiinin ilmentymistasot olivat korkeita. Endoteelisoluilla selvitettiin resistiinin vaikutuksia ateroskleroosiin liittyviin muutoksiin. Resistiini lisäsi monosyyttien kiinnittymistä endoteelisoluihin, mikä on tyypillinen ilmiö varhaiselle ateroskleroosille. Tämän työn tulosten perusteella ihmisen resistiinillä ei ole merkittävää yhteyttä insuliiniresistenssiin ja tyypin 2 diabetekseen. Sen sijaan havainto resistiinin ilmentymisestä useammissa valkosolutyypeissä, kuin mitä aikaisemmin on raportoitu, ja yhteys tulehdustekijöihin osoittaa, että ihmisen resistiinin toiminta liittyy tulehdustiloihin. Resistiini aiheuttaa myös endoteelisoluissa samanlaisia ateroskleroosille altistavia muutoksia kuin muutkin tulehdustekijät. Tulevaisuudessa plasman resistiini-pitoisuutta voidaan mahdollisesti käyttää tulehdustilojen arvioinnissa

Self-renewal and pluripotency, the hallmarks of human embryonic stem cells (hESC), confer these cells with the capacity to expand indefinitely while maintaining the ability to differentiate into any cell type of the human body; thus, making hESC a valuable source of functional differentiated cells suitable for applications in regenerative medicine, drug discovery, biotechnology, biopharmaceuticals and developmental biology. However, the large-scale production of clinical-grade hESC, required for such applications, has been hampered by the current culture conditions in which hESC still depend on the use of mouse embryonic fibroblast-conditioned medium (MEF-CM) for their efficient growth. Therefore, investigation of the factors provided by MEFs is of the utmost importance to discover which components of MEF-CM allow the long-term expansion of undifferentiated hESC. While considerable progress has been made on the identification of the protein components of MEF-CM, very little is known about the small molecules (metabolites) secreted by MEFs. In this context, an untargeted metabolomics method was developed for the investigation of potential bioactive metabolites present in MEF-CM implicated in the proliferation and/or maintenance of pluripotency of hESC in vitro. A metabolomics method was applied and successfully identified a number of metabolites which were later confirmed in their identities with the use of authentic standards, to be further investigated for their effect on hESC culture. Interestingly, the addition of PGE2, 6-keto-PGF1α, 9, 12, 13-TriHOME, 7-Ketocholesterol and stearidonic acid (the metabolites found in MEF-CM) to the unconditioned medium (UM), a medium incapable of the maintenance of hESC, showed a delay in apoptosis when compared to the negative control UM; thus, suggesting that these metabolites could help with the proliferation of hESC. Increasing evidence that hESC secrete factors into their microenvironment that can also help them to proliferate or to maintain an undifferentiated state prompted the application of the same metabolomics method to the analysis of hESC spent culture media. The results identified lysophospholipids (LPLs) as potential molecules mediating some biological activities; however, the precise role of these LPLs still remains to be determined. Overall, the results of this thesis are expected to impact and add knowledge to the field of stem cell biology providing useful information for the creation and development of more efficient and defined culture conditions for the propagation of hESC with the appropriate quality to realise their widespread application in clinic and other research areas.

Optimizing culture conditions that reduce genomic instability in human pluripotent stem cells (hPSCs) is an unmet challenge in the field. Results from our lab and numerous research groups demonstrate that hPSCs are prone to genomic aberrations and single-cell passaging increases the rate of genomic alterations. However, single-cell based passaging maintains advantages for scale-up and standardizing differentiation protocols. In this study, we investigated the problem of genomic instability in hPSC cultures with the goal towards identifying and characterizing candidate genes that could contribute to generation and survival of abnormal hPSCs. Based on microarray analysis, we identify ARHGDIA, located on 17q25, as a candidate gene conferring selective advantage to trisomy 17 hPSCs. Using lentiviral approaches to overexpress ARHGDIA in hPSCs, [hPSC (Arg)], we functionally validate that in enzymatically passaged co-cultures, hPSC (Arg) lines exhibit competitive advantage against wild type hPSCs, [hPSC (WT)]. Additionally, hPSC (Arg) lines exhibit increased single-cell survival at low density plating. In co-cultures with hPSC (WT), ROCKi exposure attenuated the competitive advantage of hPSC (Arg) subpopulations. For the first time, this work demonstrates that increased expression of a gene on 17q25 confers selective advantage to hPSCs. In parallel studies, using medium devoid of bFGF containing LIF plus two inhibitors, MEK inhibitor (PD0325901) and p38 inhibitor (SB203580), we demonstrate that hPSCs are LIF responsive and can be stably maintained in naive pluripotent culture conditions. Based on their clonal viability, we propose that naive hPSCs are a more genetically stable population than primed hPSCs, when passaged as single- cells. These studies will aid the long-term goal of hPSC scale-up while promoting stable propagation of genomically normal hPSCs.

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
The use of human primary cells provide researchers in different areas with irrefutable more biologically relevant data than using cell lines or animal blood cells. The work was performed in the scope of the Cell Biology Services @ CEDOC, aiming to provide viable and trustful human primary cells and products. We had three main objectives: protocol optimizations for blood cell isolation, culture and cryopreservation; cost estimation and divulgation of the services. We have reviewed standard protocols and compared different strategies for blood cell isolation. The impact of those methodologies was evaluated regarding cell yield and purity, cell functional characteristics and cost. We also developed a method for serum isolation from human plasma in blood buffy coats. The resultant sera were sterile and suitable to be used in leukocyte cultures. Different protocols for T cells isolation were compared: positive versus negative immunomagnetic selection and isolation using nylon wool fiber columns. Positive selection provided the highest isolation yield (32.35%), while negatively selected cells had the highest purity (92.81%). Although nylon wool fiber column was the fastest and cheapest method, unlike the immunomagnetic methods, it did not allow complete separation of T from B lymphocytes. Positive selection of monocytes was compared using two widely used commercial kits. Miltenyi’s kit provided the highest isolation yield (25.92%), recovery rate (86.70%) and purity (95.01%). Monocytes isolated with StemCell kit presented a higher cell complexity, and when differentiated into dendritic cells (DCs), showed a more mature phenotype. Differences between both kits are probably caused by the nature of the magnetic beads, suggesting caution when choosing one or other kit, as it may have an impact on DCs’ function. Overall, although dealing with apparently straight forward methodologies, our results show that testing commercial products and optimizing protocols is very important and contribute for a better quality of products and services.

Stem cell-based therapies have been proposed as promising for the maintenance, regeneration, or replacement of malfunctioning tissues, but suffer from limitations mainly due to scarce cell availability and clinical safety concern related to cell quality. Optimization of stem cell expansion process is an engineering challenge, besides a biological issue. Aim of the work presented is to develop the experimental technology and the rational insight to understand and control stem cell expansion in vitro, in terms of both average and distributed properties of the cell population produced. A rational analysis of the main phenomena involved in a cell culture was achieved, underlining the sources of stem cell heterogeneity in both conventional culture systems and stirred bioreactors. From an experimental point of view, two types of bioreactors were designed, developed, and prototyped. The first, a microliter bioreactor array, was designed based on thermoconvective mixing; this experimental setup is particularly convenient for multiparametric optimization of cell culture conditions. The second, a six-well suspension bioreactor with a working volume of 10 ml/well, was designed and fabricated for coarse process optimization or, alternatively, for small-scale stem cell production; an improved setup was developed to perform stem cell cultures under hypoxia conditions. Both devices were advantageously used to culture human cord blood-derived hematopoietic stem cells, which were then characterized according to the currently available biological assays. In order to rationally optimize the stem cell expansion process, a computational model, based on a population balance approach, was developed, that takes into account receptor and receptor-ligand complex distribution in the cell sample. The model fairly describes intrinsic intra- and inter-generational heterogeneity arising from the process of cell division. These findings could give interesting feedback to experimental design and to define the operative conditions for bioreactor cultures, in order to minimize extrinsic and intrinsic heterogeneity, and to make a step towards a clinically safe and reliable human hematopoietic stem cell expansion process.
E' stato prospettato l'impiego di cellule staminali per terapie volte al mantenimento, alla rigenerazione o alla sostituzione di tessuti malfunzionanti. Tuttavia non sono ancora state risolte alcune limitazioni legate principalmente alla scarsa disponibilità di cellule staminali e ai problemi di sicurezza clinica connessi alla qualità cellulare. L'ottimizzazione del processo di espansione cellulare è un sfida ingegneristica, oltre che biologica. Scopo di questa tesi è lo sviluppo di una tecnologia sperimentale e di un quadro razionale che consenta di comprendere e controllare l'espansione di cellule staminali in vitro, sia considerando le proprietà medie che la loro distribuzione nella popolazione cellulare prodotta. E' stata realizzata un'analisi razionale dei principali fenomeni coinvolti nella coltura cellulare, ponendo in evidenza le fonti di eterogeneità sia nei sistemi di coltura convenzionali che nei bioreattori mescolati. Da un punto di vista sperimentale, sono stati progettati e sviluppati due tipi di bioreattori fino a realizzarne dei prototipi. Il primo, un sistema di bioreattori di volume dell'ordine dei microlitri, è stato progettato basato su un meccanismo di termoconvezione; questo apparato sperimentale è particolarmente adatto per un'ottimizzazione multiparametrica delle condizioni di coltura. Il secondo, un bioreattore in sospensione multipozzetto con un volume operativo di 10 ml/pozzetto, è stato pensato e costruito per un'ottimizzazione di processo meno dettagliata o, alternativamente, per una produzione su piccola scala di cellule staminali; una versione più avanzata è stata sviluppata per effettuare colture di cellule staminali in condizioni di ipossia. Entrambi i dispositivi sono stati vantaggiosamente utilizzati per coltivare cellule staminali ematopoietiche, ricavate da cordone ombelicale umano, che sono poi state caratterizzate secondo i metodi di analisi biologica convenzionali. Per ottimizzare razionalmente il processo di espansione delle cellule staminali, è stato sviluppato un modello computazionale, basato su un bilancio di popolazione, che tiene conto della distribuzione di recettori e di complessi recettore-ligando nel campione cellulare. Il modello descrive ragionevolmente l'eterogeneità intrinseca, intra- e intergenerazionale, derivante dal processo di divisione cellulare. Questi risultati possono dare un riscontro positivo in fase di progettazione degli esperimenti e di definizione delle condizioni operative a cui effettuare colture in bioreattore, al fine di minimizzare l'eterogeneità estrinseca e intrinseca della popolazione cellulare e di effettuare un ulteriore avanzamento verso un processo di espansione di cellule staminali umane clinicamente sicuro ed affidabile.

This research addresses the conflictualization dynamics induced by the politicization of religion at the supranational level. It tests the Union’s institutional capacity to routinize dissension, temper animosities, and reconcile divergences in the light of religiously-loaded, issue-specific controversies. So-called “morality issues” such as abortion or human embryonic stem cell research emerged onto the EU agenda at the end of the 1990s. The main sites of bioethical contention correspond to the European Parliament and Commission arenas, where political, social, and religious entrepreneurs have been pursuing ideological interests of either liberal or conservative nature. We developed an interpretative approach to their discourses and perceptions through the qualitative content analysis of semi-structured interviews and online documentary sources. A key task consisted in determining whether one observes the routinization of bioethical conflicts by European institutions (“business-as-usual” scenario) or whether the said conflicts are remaining extrinsic to the Brussels political game (“culture wars” scenario). In other words, is the emergence of new stakes – morality issues – prompting the emergence of new divisions and repertoires of action? We put to the test the normal course of EU politics in the light of (1) the structuration of morality divides along religious, political, and national frontlines; and (2) the materialization of morality antagonisms through discursive, bureaucratic, and mobilization weapons. Overall, the “polarization” and “political style” variables showed that the supranational debates on abortion and hESCR do not fully alter the logics of supranational governance; in return, the EU polity is not closed to the crystallization of politicized modes of dissent expression. The hypothesis of an intermediary scenario oscillating between policy-seeking and position-taking perspectives is thus confirmed. On the one hand, issue-specific alliances characterized by internal multifold diversities do play the institutional rules of the European political game in their quest for ideological influence on the decision-making process. On the other hand, limited supranational competences on religiously-loaded issues constrain conflicting factions’ leeway to a symbolic use of morality causes and beliefs as instrumental devices worth of credit-claiming and identity-posturing.
Doctorat en Sciences politiques et sociales
info:eu-repo/semantics/nonPublished

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Bachelors
Burnett College of Biomedical Sciences
Molecular and Microbiology

The zygote and blastomeres of cleavage stage mouse embryos have the capacity to differentiate to the embryonic and both extra-embryonic lineages and are considered functionally totipotent. Until now, it has not been possible to establish stable cell lines that resemble these totipotent-like cells. In this work, I hypothesised that by modulating signalling pathways known to be important in early embryonic development it may be possible to capture in vitro a self-renewing state that possessed features of pre-implantation blastomeres. I succeeded in formulating a novel hypothesis driven cell culture medium which allowed the establishment of a stem cell state that possessed expanded differentiation potential to the embryonic and both extra-embryonic lineages in vitro and in vivo. These cells were isolated directly from in vitro culture of mouse pre-implantation embryos or single cell blastomeres, reprogrammed from somatic cells or converted from mouse ES cells. With these cells, I generated single cell chimeras which demonstrated extensive contribution to all lineages in the developing organism providing additional evidence that this chemical medium maintained a homogenous stem cell population. I demonstrated that the transcriptome of these cells was enriched with an early pre-implantation blastomere signature, distinct from other rare published totipotent-like cells. Finally, I demonstrated that the same chemical formulation permitted the establishment in vitro of a human stem cell state that possessed expanded differentiation potential to the embryonic and extra-embryonic lineage in vitro. My work has shown for the first time that through chemical modulation of pathways implicated to be involved in pre-implantation development, a novel homogenous stem cell state that possesses a pre-implantation transcriptional signature and expanded differentiation potential to both the embryonic and extra-embryonic lineage can be established and maintained in vitro in both mouse and human, suggesting a possible interspecies conservation of the signalling networks involved in early embryonic development.

The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient's healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker a- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP. These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

The limbal stem cell population is located in the limbal junctional zone between the cornea and the conjunctiva, and is responsible for maintaining the corneal epithelium. Damage to the limbal stem cell population results in a condition known as limbal stem cell deficiency (LSCD), which is characterised by conjunctivalisation of the cornea, visual impairment and persistent irritation. To treat LSCD, an alternative source of human limbal epithelial (HLE) cells must be transplanted back onto the diseased cornea. Limbal tissue grafts have had a moderate degree of success. However, autologous grafts risk damage to the healthy eye, whilst allogeneic grafts are susceptible to immunological rejection. Cultured HLE grafts offer a promising alternative to whole tissue grafts. The production of cultured HLE grafts involves the removal of a small (1-2 mm2) biopsy from the patient’s healthy limbus, followed by ex vivo expansion to produce an epithelial sheet, which is subsequently transplanted onto the damaged corneal surface. However, the production of cultured HLE grafts usually requires the addition of animal-derived products during cell culture. Animal-derived components, such as foetal bovine serum (FBS) and murine 3T3 feeder cells, introduce the patient to potential crossspecies infection and immune responses to xenogeneic antigens. Consequently, the overall aim of this project has been to develop a culture technique free of xenogeneic products for the establishment and propagation of HLE cells. To achieve this aim, alternatives to FBS in the culture medium and 3T3 feeder cells were pursued. A defined serum-free medium (SFM) containing vitronectin (VN), insulin-like growth factor binding protein 3 (IGFBP3), insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) was investigated as an alternative to serumsupplemented medium (SSM) for HLE cell culture. Initial studies focused on the effects of these growth factors on HLE cell metabolic activity and migration. Metabolic activity was primarily stimulated by IGF-I and EGF, with the combination of IGF-I and EGF in solution stimulating metabolic activity to a significantly greater extent than the SSM positive control (p = 0.006). HLE cell migration was also effected by combinations of VN, IGFBP3, IGF-I and EGF. Migration was stimulated above the SFM negative control by the combination of IGFBP3 and IGF-I either with or without the addition of EGF. However, the presence of VN was required for optimal migratory responses (p < 0.003). Hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) were also investigated as additional components to the SFM formulation. HGF significantly stimulated HLE cell metabolic activity and migration (p < 0.02). In contrast, KGF did not significantly stimulate either HLE cell metabolic activity or migration. The addition of either HGF or KGF to the SFM supplemented with VN, IGFBP3, IGF-I and EGF did not significantly enhance the metabolic activity of HLE cells. Therefore, HGF and KGF were no longer pursued as additional components to the SFM formulation. Additional studies were conducted to examine the efficacy of replacing murine 3T3 feeder cells with human ocular stromal cells during HLE cell culture. Initially, stromal cells were isolated from the cornea, limbus and sclera to determine whether there were differences between these stromal cell populations. The results indicated that scleral stromal cells had a significantly larger area and perimeter than either corneal or limbal stromal cells (p < 0.001). Scleral stromal cells were also significantly more rounded than either corneal or limbal stromal cells, as determined by the elliptical factor equation (p < 0.001). Immunocytochemistry also revealed that scleral stromal cells expressed significantly more of the myofibroblast marker ..- smooth muscle actin than either corneal or limbal stromal cells (p < 0.001), and significantly less of the fibroblast/myofibroblast marker Thy-1 than corneal or limbal stromal cells (p < 0.001). Therefore, scleral stromal cells were identified as different in comparison to corneal and limbal stromal cells. Primary HLE cells were cultured with irradiated corneal, limbal and scleral stromal cells. HLE cultures established with either corneal or limbal stromal feeder cells contained more cellular protein (as measured by rhodamine B dye absorbance) than cultures established without feeder cells (p < 0.001). The colony forming efficiency (CFE) of HLE cells established with corneal or limbal stromal feeder cells was also significantly greater than HLE cells established without feeder cells (p < 0.001). In contrast, HLE cultures established with scleral stromal feeder cells contained low levels of cellular protein and had a low CFE, which was not significantly different to the HLE cultures established without feeder cells. Immunocytochemistry indicated that HLE cultures established with scleral feeder cells also showed lower expression of the stem cell markers ABCG2 and C/EBP ... These results suggest that freshly isolated HLE cells can be cultured with irradiated corneal or limbal stromal cells as a replacement for murine 3T3 feeder cells. Finally, the SFM supplemented with VN+IGFBP3+IGF-I+EGF was combined with limbal stromal feeder cells, and examined as a culture technique free of animalderived products. Freshly isolated HLE cells established in SFM supplemented with VN+IGFBP3+IGF-I+EGF and limbal feeder cells contained a similar amount of cellular protein (as measured by crystal violet dye absorbance) when compared to the SSM+3T3 positive control. In addition, the CFE of freshly isolated HLE cells established in VN+IGFBP3+IGF-I+EGF and limbal feeder cells was significantly higher than the SSM+3T3 positive control (p = 0.004). However, a live/dead assay revealed a reduced HLE cell viability in SFM supplemented with VN+IGFBP3+IGFI+ EGF and limbal feeder cells after seven days in culture. In addition, immunocytochemistry demonstrated a lower expression of the stem cell markers ABCG2 and C/EBP .. in the SFM treatment with limbal feeder cells. Therefore, freshly isolated HLE cells can be cultured in SFM supplemented with VN+IGFBP3 +IGF-I+EGF and limbal feeder cells. However, this culture technique is less likely to support the growth of immature limbal stem cells when compared to the SSM+3T3 positive control. Overall, this research has attempted to create a culture system free of animal-derived products for the production of cultured HLE grafts to treat limbal stem cell deficiency. The results show that HLE cells respond to a serum-free medium formulation containing VN+IGFBP3+IGF-I+EGF. In addition, this culture medium can be combined with irradiated stromal cells isolated from the limbus to support HLE culture production. However, the combination of VN+IGFBP3+IGF-I+EGF and limbal feeder cells demonstrated a reduced viability, which indicates that further refinement of the formulation is required. This thesis has also demonstrated differences between stromal cells isolated from the cornea, limbus, and sclera, and has generated knowledge which may impact on the understanding of stromalepithelial regulation.

Background: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. Methods: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. Results: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. Conclusions: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Amniotic fluid stem cells (AFSCs) offer therapeutic potential for prenatal and neonatal diseases based on their unique features. From the development of embryos, AFSCs represent a category between embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs), with AFSCs more primitive than MSCs. Our lab has previously established that AFSCs can be reprogrammed to regain functional pluripotency with valproic acid (VPA). However, detailed mechanisms are still unknown. Here, our results showed that Wnt signalling was downregulated in the initial stage and upregulated with VPA treatment; whereas mesenchymal-to-epithelial transition (MET) was not observed in the process. Additionally, our previous results demonstrated that AFSCs maintained in ESC conditions shared 82% transcriptome similarity with ESCs. In the second part of this study, we revealed that features of AFSCs including marker expression and differentiation ability were sustained better in ESC conditions. Regarding osteogenesis, enhanced osteogenic ability was found in AFSCs maintained in ESC conditions due to a TGF-beta/CD73-dependent signalling pathway. Moreover, in addition to possessing the same tri-lineage differentiation capability as MSCs, AFSCs can also be induced to express cardiac markers, but contractile cells have not been obtained yet. As features of AFSCs are better preserved in ESC conditions, a Wnt-dependent cardiomyocyte differentiation protocol for pluripotent stem cells is examined on AFSCs in the last part of this study. Our results showed that, with the Wnt-dependent protocol, cardiac markers were induced but spontaneously contractile cells were not observed. Taken together, our findings show that (1) Wnt signalling may play a role in VPA-induced reprogramming, (2) AFSCs maintained in ESC conditions can better maintain stem cell features, especially osteogenic ability through a TGF-beta/CD73 pathway, (3) With a Wnt-dependent protocol, AFSCs can be induced to express cardiac markers but not to become contractile cells.

Dissertation to obtain master degree in Genética Molecular e Biomedicina
Information currently available on neurotoxicity of chemicals is scarce and there are a growing number of new compounds to be tested. Therefore, new strategies are necessary to identify neurotoxic agents with speed, reliability and respect for animal welfare. The limited availability of primary human brain cells means that there is a need for human cell lines that reliably model human neurons and astrocytes. Despite the advances in stem cell research, numerous challenges must be overcome before this technology can be widespread used, such as low differentiation efficiency. Human pluripotent embryocarcinoma NTera2/cloneD1 (NT2) cell line is an alternative cell source from which neurons and astrocytes can be derived in vitro. The aim of this work was to develop scalable and reproducible novel human cellular models using NT2 cells as source of differentiated neural phenotypes. A 2D culture system for astrocytic differentiation was implemented. After 4 weeks of differentiation with retinoic acid followed by 5 weeks maturation with mitotic inhibitors, astrocytes obtained expressed vimentin, GFAP, S100- and GLT-1 as characterized by immunodetection and qRT-PCR. Then, a 3D culture approach was adopted, using stirred suspension culture systems, in which cell-cell and cell-extracellular matrix interactions occur, mimicking better the in vivo situation. NT2 cells, inoculated as single cells, spontaneously aggregated without compromising their pluripotency. Optimization of stirring rate allowed control of aggregate size along time. After 3 weeks of RA treatment and 2 weeks of maturation, neurons expressing βIII-tubulin, MAPs and synaptophysin and astrocytes expressing vimentin, GFAP, S100- and GLT-1 were detected, as characterized by immunodetection and qRT-PCR. Furthermore, astrocytes presented a 2.5-fold higher yield than that observed in 2D culture systems. Results showed that NT2 differentiated cells are promising models for neurotoxicity testing. Furthermore, the 3D culture systems developed herein can contribute to increase the relevance of these studies, recapitulating human neuron-astrocyte interactions in a 3D cellular context.
Fundação para a Ciência e Tecnologia - PTDC/EEB-BIO/112786/2009

Among all the possible sources of mesenchymal stem cells, adipose tissue and olfactory mucosa have raised great interest and have become some of the most investigated sources. Adipose tissue-derived mesenchymal stem cells and the fat itself as a source of human adipose derived stem cells, represent one of the major fields of research in regenerative medicine. A great advantage is represented by the minimal invasive and high accessibility to adipose tissue and its ready availability. In the present study, hADSCs were isolated from the adipose tissue donated by several patient and have been investigated and characterized through different technical approaches, such as flow cytometry and immunocytochemistry. These hADSCs reproducibly fulfill the general definition of MSCs by both phenotypic and differentiation capabilities criteria, showing also the expression of neural markers, as observed by confocal microscope analysis. Lipoaspirated adipose tissue showed positivity to ß-tubulin III that was also maintained in lipoaspirate-derived hADSCs. A population of stem cells retaining typical characteristics of surface markers of classical adipose tissue stem cells and MSC was obtained when adipose tissue was subjected to culture in vitro, either by processing through centrifugation or by direct plating without enzymatic digestion with collagenase. Flow cytometry analyses showed that hADSCs expressed classical mesenchymal markers such as CD44, CD73, CD90, CD105 and CD166, while endothelial (CD31, CD34, CD144, CD146) and hematopoietic (CD45, CD133) markers were much less represented. Also the ability to give rise to tissue of mesenchymal origins, such as osteoblastic and adipogenic lineages, were present in hADSCs. In addition, the immunofluorescence staining indicated the expression of neural stem markers in hADSCs which consequently co-expressed nestin, β-tubulin III and glial GFAP. We have also characterized human olfactory ensheathing stem cells. Olfactory mucosa is specialized tissue inside the nasal cavity involved in olfactory perception and capable of lifelong regeneration throughout adulthood. Multipotent stem cells obtained from it offer the possibility of promoting regeneration and reconstruction in regenerative medicine, being readily accessible with minimal invasive techniques, capable of expansion in vitro and retaining broadly potent differentiative capacity as stem cell progenitors. Among the several members of the olfactory mucosa, Olfactory Ensheathing Cells (OECs) are well known to be useful in repairing the nervous system. By following our method, cells can be easily isolated and maintained in TCM, and their cultivation in large flasks allowed obtaining rich cultures of OECs in 2 weeks. Cell cycle analysis showed that the majority of cells are in G0/G1 phase, while just a lesser part is in S/G2 phase. In our growth conditions, no chromosomal abnormalities were observed also at high culture passage (p14). Live morphology of obtained cells showed a fibroblast-like phenotype and the immunohistochemical analyses showed the expression of beta-Tubulin III, Vimentin, Nestin, Glial Fibrillary Acidic Protein and Microtubule-Associated Protein 2. By FACS analysis we demonstrated that OECs are positive to typical surface mesenchymal markers (CD44, CD73, CD90, CD105, CD146 and CD166). As expected, some endothelial (CD31, CD34) and hematopoietic (CD45) markers were very few represented, while some others (CD56, CD144, CD146, CD133) are partially found. These cells also express genes that constitute the core circuitry of self-renewal such as SOX2, NANOG and OCT4 and the stemness marker CD133. OECs incubated with serum-free medium, normally used for the formation of neurospheres, spontaneously formed large spheroids reaching a mean diameter of 100 μm in 10 days of culture. Immunofluorescence of specific proteins showed that spheres were positive to markers such as Nestin, Vimentin, TUJ-1, MAP2 and GFAP. In conclusion, our method allows the quickly and easily hADSCs and hOESCs isolation from human adipose tissue and nasal biopsies. The obtained cells can be cultured without altering their mesenchymal properties, suggesting the pluripotency nature of these cells and that they are a reliable source for regenerative medicine.

Autologous explant-derived cardiac stem cell (EDC) therapies are a promising therapy for ischemic cardiomyopathy, but straightforward clinical translation is limited by traditional culture conditions which are often supplemented with ill-defined and xenobiotic components such as fetal bovine serum. Therefore, we investigated the influence of a commercially sourced serum-free (SF) xenogen-free medium on human EDC yield, phenotype, in vitro measures of EDC performance, and post-infarct cardiac repair using an immunodeficient mouse model of acute myocardial infarction. Despite reduced production of several pro-cardiogenic cytokines, SF EDCs promoted similar vessel formation, circulating stem cell recruitment and cardiogenic differentiation as compared to standard cultures. Transplant of SF EDCs into immunodeficient mice 1 week after myocardial infarction boosted post-ischemic repair beyond that of standard EDCs by enhancing viable myocardium within the infarct. These findings demonstrate that serum-free culture methods provide a superior cardiac-derived cell product with ready clinical translatability.

Master of Science
Department of Grain Science and Industry
X. Susan Sun
Human induced pluripotent stem cells (hiPSCs) are generated from human somatic cells using defined transcription factors. These cells possess characteristics very similar to that of human embryonic stem cells including the ability to differentiate into cell types of all three germ layers. HiPSCs show great potential in clinical researches like drug screening and regenerative medicine, that all require large amount of cells cultured under well-defined conditions. The most common culture methods used for hiPSCs are 2D culture methods using Matrigel or vitronectin coated culture plates or flasks. 2D culture methods require large surface area to produce the same amount of cells compared to 3D methods. In addition, cells cultured in 2D culture environment are far from that in vivo. In this study, we developed a robust 3D culture condition based on hiPSC-qualified PGmatrix (PGmatrix-hiPSC) hydrogel. This 3D culture system provide hiPSCs with well-defined, more in vivo-like environment that encapsulate cells in liquid rich hydrogel with appropriate oxygen supply that resembles the hypoxia condition in vivo. Two hiPSC lines grown continuously in PGmatrix-hiPSC showed higher total population expansion and higher viability, with more consistency compared to the same cell lines grown in 2D on Matrigel or Vitronectin-XF. After grown in 3D PGmatrix-hiPSC for over 25 passages, major pluripotency markers, such as Oct4, Sox2, Nanog, and SSEA4 are expressed in most hiPSCs examined by flow cytometry. RT-qPCR also confirmed adequate expression levels of major pluripotency related genes. In addition, karyotype analysis of hiPSC after 37 passages in 3D PGmatrix-hiPSC was found normal. The same hiPSC lines cultured continuously in parallel in 2D and 3D showed differences in gene expression and surface marker TRA-1-81 expression. These results indicated the 3D PGmatrix-hiPSC system is likely superior in maintaining hiPSC growth as well as pluripotency. The findings also suggest that it is very important to study cells in 3D culture environment to better understand the mechanism of pluripotency maintenance.

The ovary, one of the most active endocrine organs in human body, plays active rather than passive roles in the initiation and maintenance of the reproductive cycle. Responding to cyclic pituitary gonadotropin secretion, the various compartments of ovary interact in a highly integrated manner to produce a fertilizable ovum and secrete sex steroid hormones regulating the menstrual cycle and maintaining female secondary sexual characteristics. The ovary is the major source of female hormone production. The steroidogenic activity in human ovary has been reported to start at the stage of embryonic development when the fetal ovary was found to possess not only cholesterol side-chain cleavage activities but also 17a-hydroxylase as well as 17-20 desmolase activities (Adashi, 1994). Through each menstrual cycle, the ovary has been documented to produce a variety of classic hormones and growth factors. Each hormone is actively involved in regulating different aspects of ovarian function via different mechanisms. Our knowledge about hormonal regulation in menstrual disorders, infertility and miscarriage is still not complete. It is not clear if other hormones or growth factors are involved in the regulation of human menstrual cycle. Tissue inhibitor of metalloproteinase-l (TIMP-l) is traditionally considered in its role as a metalloproteinase inhibitor regulating tissue remodelling. However, studies have found that TIMP-1 exhibits a series of other biological functions such as binding with procathepsin L to stimulate steroidogenesis (Boujrad et al., 1995), promoting cell growth (Hayakawa et al., 1992), matrix binding, inhibiting angiogenesis (Moses et al., 1990) and inducing apoptosis (Brew et al., 2000, Thorgeirsson, 1996). All these effects of TIMP-1 suggest a number of different mechanisms by which TlMP-l could be involved in the life cycle of the ovarian follicle and corpus luteum. As the general aim of this study is to investigate the role of TIMP-1 as a novel factor in regulating ovarian cycle and steroidogenesis by ovarian cells, the focus of this review will be on the ovarian cycle, and the production and regulation of hormones during ovarian cycle, particularly the existing knowledge about TIMP-1.

T cell development is regulated by signals generated in the interactions between developing thymocytes and the thymic stroma. Using fetal thymus organ culture (FTOC) as a model of T cell development, we investigated the ability of two potent signal modulators to influence this process. These studies show that both nicotine and tumor necrosis factor-alpha have the ability to influence T cell receptor (TCR) signaling and the maturational capacity of treated cultures. FTOC treated with low concentrations of nicotine produced more immature T cells and fewer mature T cells. These expanded populations of cells also expressed CD69, CD95 (FAS) and elevated levels of recombinase activating genes (RAG). This phenotype reflects the fact that these cells have received a positive selection signal, are for apoptosis and are likely attempting secondary TCR rearrangements. Nicotine effects were partially blocked by the nicotinic antagonist, d-tubocurarine. Furthermore, d-tubocurarine alone blocked the development of T cells entirely, suggesting the presence of an endogenous ligand that may engage nicotinic acetylcholine receptors and regulate normal thymopoeisis. These observations underscore the linkage between the nervous and the immune systems, not only in terms of shared resources, but also in terms of direct interactions between these two systems. In another study we used FTOC and an associated in vitro Type 1 diabetes mellitus model to reconcile the role of TNF-alpha in thymopoiesis with its role in diabetes. Our data indicate that thymocytes from NOD FTOC express lower levels of TNF receptors and produce more TNF-alpha compared to non-diabetic C57BL/6 (B6) FTOC. Neutralization of endogenous TNF-alpha in NOD FTOC with a soluble TNF receptor (sTNF R1) rescued insulin production in our in vitro diabetes model. NOD FTOC treated with TNF-alpha produced greater numbers of mature T cells and a higher percentage of cells expressing CD95L (Fas ligand). Treatment with sTNF R1 had the opposite effect. TNF-alpha's known ability to attenuate TCR signaling coupled with these observations suggest that its overproduction in these animals may be driving T cells to maturity, altering the process of negative selection and ultimately enhancing the survival of potentially diabetogenic T cells resulting in disease susceptibility in these animals.